Embryonic stem cells are promising candidates for a source of cells for cell transplantation for Parkinson's disease and diabetes mellitus. However, even embryonic stem cells derived from mice, humans and other primates require the coexistence with mouse-derived feeder cells (stromal cells) in their culture and differentiation induction, and this represents a major barrier against their clinical applications.
Recently, the present inventors developed a method of inducing the differentiation of mouse and monkey embryonic stem cells into neurons at high efficiency (SDIA method) (see pamphlet for International Patent Publication No. WO01/088100; pamphlet for International Patent Publication No. WO03/042384; Kawasaki et al., Neuron, vol, 28, p. 31-40 (2000); Kawasaki et al., Proceedings of the National Academy of Sciences of the USA, vol, 99, p. 1580-1585 (2002); Mizuseki et al., Proceedings of the National Academy of Sciences of the United States of America, vol, 10, p. 5828-5833 (2003); Ooto et al., Invest. Ophthalmol. Vis. Sci., vol, 44, p. 2689-2693 (2003)). Using this method, the present inventors succeeded in producing in vitro dopamine-secreting neurons, which are expected to be applied to transplantation therapy for Parkinson's disease, and motor neurons, which are speculated to be applied to treatment for amyotrophic lateral sclerosis, from mouse and monkey embryonic stem cells (see pamphlet for International Patent Publication No. WO01/088100; pamphlet for International Patent Publication No. WO03/042384; Kawasaki et al., Neuron, vol, 28, p. 31-40 (2000); Kawasaki et al., Proceedings of the National Academy of Sciences of the USA, vol, 99, p. 1580-1585 (2002); Mizuseki et al., Proceedings of the National Academy of Sciences of the United States of America, vol, 100, p. 5828-5833 (2003); Ooto et al., Invest. Ophthalmol. Vis. Sci., vol, 44, p. 2689-2693 (2003)).
However, considering human applications in a long viewpoint, one of the major problems with this SDIA method is the necessity for coculture with embryonic stem cells during differentiation induction of mouse bone marrow-derived stromal cells (PA6 cells). To date, differentiation of embryonic stem cells into nerves has not efficiently been performed in the absence of stromal cells such as PA6 cells. However, because of coculture with mouse-derived cells, even neurons produced using human embryonic stem cells involve the same level of risk as heterologous transplantation with regard to the safety of transplantation (in terms of contamination with heterologous cells and pathogens, and the like), thus posing a major barrier against their clinical applications.
Also, as another differentiation-inducing method for embryonic stem cells, a method using retinoic acid is known (see Bain et al., Dev. Biol., vol, 168, p. 342-357 (1995)), but this method has been problematic in that the dopamine nerve differentiation induction rate is very low. Also, although a method of selection culture of nerve precursor cells differentiated from aggregation cultured ES cells using a selection medium has also been known (see Lee et al., Nature Biotech., vol, 18, p. 675-679 (2000)), this method has been problematic in that it takes a time doubling to tripling that for the SDIA method.
Against this background, there has been a strong demand for a method that assures the safety of transplantation of cells obtained by culturing embryonic stem cells and also enables efficient induction of differentiation of embryonic stem cells into particular functional cells, for example, neurons.